Iron is an essential trace element that is required for growth and development of all living organisms. It is indispensable for DNA synthesis and is an essential component of many proteins and enzymes including haemoglobin and myoglobin, the cytochromes, NADH dehydrogenase, lipooxygenases, phosphatases, superoxide dismutase, ribonucleotide reductase, and fatty acid desaturases.
Iron can also be toxic when present in excess because of its ability to generate reactive oxygen species. This dual nature imposed a very tight regulation of the iron concentration in the body. Disturbances in iron metabolism are implicated in a number of significant human diseases, including anemia of chronic diseases, anemia of inflammation, or the iron overload disease hemochromatosis.
In mammals, iron absorption occurs predominantly in the duodenum and upper jejunum, and systemic iron homeostasis is regulated at the level of intestinal absorption and this is the only mechanism by which iron stores are physiologically controlled (Philpott, Hepatology 35:993-1001 2002). Following absorption, iron is bound to circulating transferrin and delivered to tissues throughout the body. The liver is the major site of iron storage. There, transferrin-bound iron is taken into the hepatocytes by receptor-mediated endocytosis via the classical transferring receptor (TfR1) (Collawn et al., Cell 63: 1061-1072, 1990) and presumably in greater amounts via the recently identified homologous transferrin receptor 2 (TfR2) (Kawabata et al., J. Biol. Chem. 274: 20826-20832, 1999). The extracellular domain of this protein is 45% identical to the corresponding portion of TfR1 (Id.). TfR2 can also bind diferric transferrin and facilitate the uptake of iron. Mutations in TfR2 have been associated with certain forms of hemochromatosis demonstrating the important role for TfR2 in iron homeostasis (Philpott, Hepatology 35:993-1001, 2002; Camasehella et al., Nat. Genet. 25:14-15, 2000; Fleming et al. Proc. Natl. Acad. Sci. USA 99: 10653-10658, 2002). TfR2 is predominantly expressed in the liver (Fleming et al., Proc. Natl. Acad. Sci. USA 97: 2214-2219, 2000; Subramaniam et al., Cell Biochem. Biophys. 36:235-239, 2002), and is localized in the basolateral membrane domain of hepatocytes. (Merle et al., Histochem. Cell. Biol., 2006.)
Maintenance of stable extracellular iron concentrations requires the coordinate regulation of iron transport into plasma from dietary sources in the duodenum, from recycled senescent red cells in macrophages and from storage in hepatocytes.
Hepcidin is a recently discovered peptide hormone (Park et al., J. Biol. Chem. 276:7806-7810, 2001; Krause et al., FEBS Letter 480:147-150, 2000), which is the key regulator of systemic iron homeostasis. Hepcidin is predominantly produced in the liver (Park et al. J. Biol. Chem. 276:7806-7810, 2001; Kulaksiz et al. GUT 53:735-43, 2004), circulates in plasma and is excreted in urine (Kulaksiz et al., J. Endocrinol. 184, 2005). It is encoded by a small three-exon gene as a preprohepcidin with a characteristic signal sequence and a furin cleavage site preceding the mature hepcidin peptide. The active form of the peptide is a 25 amino acid β-sheet hairpin stabilized by four disulfide bonds. It is synthesized as a preprohepcidin of 84 amino acids. The signal peptide is cleaved leading to the 60 amino acids prohepcidin, which is further processed giving rise to the 25 amino acids hepcidin. In human urine, the predominant form is the 25 amino acid peptide, although shorter peptides with 20 and 22 amino acids are also detectable.
The involvement of hepcidin in iron metabolism was suggested by the observation that hepcidin synthesis is induced by dietary iron (Pigeon et al. J. Biol. Chem. 276:7811-7819, 2001). The specific role of hepcidin was then examined by assessing the effects of its deficiency or excess in transgenic mouse models. Hepcidin expression is abolished in mice exhibiting iron overload due to targeted disruption of the upstream stimulatory factor 2 (Usf2) gene, resembling the same phenotype as found in hfe−/− mice (Nicolas et al., Proc. Natl. Acad. Sci. USA 98:8780-8785, 2001). In contrast, overexpression of hepcidin was shown to result in severe iron deficiency anemia in transgenic mice (Nicolas et al., Proc. Natl. Acad. Sci. USA 99:4396-4601, 2002), indicating that hepcidin is a central regulator of iron homeostasis. Moreover, recent studies have shown that liver hepcidin expression is decreased in the hfe knockout mouse (Ahmad et al., Blood Cells Mol. Dis. 29, 2002), and mutations in the hepcidin peptide are associated with severe juvenile hemochromatosis (Roetto et al., Nat. Genet. 33, 2003), providing new perspectives in our understanding of the molecular pathogenesis of iron overload.
Recent studies indicate that hepcidin inhibits cellular efflux of iron by binding to ferroportin (Nemeth et al. Science 306: 2090-2093, 2004), the only known mammalian iron exporter, which is expressed by enterocytes, macrophages and hepatocytes. The binding of hepcidin causes ferroportin to be internalized and degraded, and the loss of ferroportin from cell membrane ablates cellular iron export. The direct hepcidin-ferroportin interaction allows an adaptive response from the body in situations that alter normal iron homeostasis (hypoxia, anemia, iron deficiency, iron overload, and inflammation).
This mechanism explains the regulation of iron absorption. When iron stores are adequate or high, the liver produces hepcidin which circulates to the duodenum, where hepcidin causes internalization of ferroportin, blocking the sole pathway for the transfer of iron from the enterocytes to plasma. When iron stores are low, hepcidin production is suppressed, and ferroportin molecules are displayed on basolateral membranes of enterocytes, transporting iron from enterocyte to plasma (Ganz, Best Prac. & Res. Clin. Haem. 18, 2005). Most of the iron absorbed from the diet or recycled from haemoglobin is destined for developing erythrocytes. It is therefore not surprising that hepcidin production is homeostatically regulated by anemia and hypoxemia (Nicolas et al., J. Clin. Invest. 110, 2002). When oxygen delivery is inadequate, the homeostatic response is to produce more erythrocytes. Thus, in anemia, hepcidin levels decrease, its inhibitory effects diminish, and more iron is made available from the diet and from the storage pool in macrophages and hepatocytes.
Hepcidin, as an iron-regulatory hormone, constitutes an important link between host defense, inflammation and iron metabolism. Hepcidin is structurally similar to cysteine rich, cationic, antimicrobial peptides, including the defensins and some cathelicidins. In vitro, human hepcidin exerts antimicrobial and antifungal activities (Park et al., J. Biol. Chem. 276:7806-7810, 2001; Krause et al., FEBS Letter 480:147-150, 2000). Its synthesis is markedly induced by infection and inflammation (Pigeon et al., J. Biol. Chem. 276: 7811-7819, 2001; Nemeth et al., 101, Blood 2003; Nicolas et al., J. Clin. Invest. 110, 2002), trapping iron in macrophages, decreasing plasma iron concentrations and causing iron-restricted erythropoiesis characteristic of anemia of inflammation.
The cytokine IL-6 is apparently the key inducer of hepcidin synthesis during inflammation (Nemeth et al., J. Clin. Invest. 113, 2004) and anti-IL-6 antibodies block the induction of hepcidin mRNA in human hepatocyte cell lines treated with supernatants of LPS- or peptidoglycan-stimulated macrophages. During inflammation induced by subcutaneous injections of turpentine, normal mice show a marked decrease in serum iron (Nicolas et al., J. Clin. Invest. 110, 2002; Nemeth et al., J. Clin. Invest. 113, 2004). This response is completely ablated in hepcidin-deficient mice and in IL-6-deficient mice. In humans, the hepcidin increase elicited by IL-6 infusion is accompanied by a decrease in serum iron and transferring saturation of more than 30% (Nemeth et al., J. Clin. Invest. 113, 2004; Ganz, Best Prac & Res Clin Haem 18, 2005).
The key role of hepcidin in iron homeostasis and its disorders suggests that its assay in blood or urine could prove useful for the diagnosis and monitoring of iron disorders. Furthermore, hepcidin could be a marker for disease activity of chronic inflammatory diseases such as, for example, chronic polyarthritis or Crohn's disease, or ulcerative colitis. At this time, only an assay for pro-hepcidin is available (Kulaksiz et al., GUT 53: 735-43, 2004; see also co-pending related U.S. patent application Ser. No. 10/441,089, filed May 19, 2003 and Ser. No. 10/299,486, filed Nov. 19, 2002). Further development of reliable plasma and urine assays for hepcidin is highly desirable (Ganz, Best Prac. & Res. Clin. Haem. 18, 2005). However, production of specific antibodies against hepcidin was not possible due to the complicated structure of hepcidin (Ganz, Best Prac. & Res. Clin. Haem. 18, 2005; Kulaksiz et al., GUT 53: 735-43, 2004; Hugman, Clin. Lab. Haem. 28, 2006).
Thus, there is a need for improvements in methods and kits useful for the diagnosis and monitoring of disease conditions characterized by non-physiological levels of hepcidin protein.